SUMMARY Theoverallgoalofthisresearchistoprovideaflexibleprenatalgenetictestingkitthatcanbeexpandedtodetect anyinheritabletraitasearlyas5,andupto20,weeksofgestation,fromasafe,noninvasivePapsmear.Studies showthatperinatalPaptestsposenorisktomotherorfetus,andcapturetrophoblast-likecellsthatmigratefrom the placenta into the reproductive tract. Trophoblast retrieval and isolation from the cervix (TRIC) efficiently isolateshundredsoffetalcellswithoutlimitationsduetoearlygestationalage,maternalobesity,oruteroplacental insufficiencydisorders.InarecentScienceTranslationalMedicinereport,weisolatedsufficientgenomicDNA fromintactfetalcellsobtainedbyTRICat5-19weeksofgestation(n=20)todefinitivelydistinguishmaternaland fetal DNA by targeted next-generation sequencing (NGS) of 59 short terminal repeats (STRs) and 94 single nucleotide polymorphisms (SNPs). Compared to massively parallel sequencing of cell-free fetal DNA from maternalserum,whichhasafetalfractionofonly4-10%atweek10ofgestation,DNAobtainedbyTRIChada fetalfractionof85-100%,capableofprovidingnucleotide-specifichaplotyping.TRICwillbecommercializedto identifysinglegeneandchromosomenumberdisordersprenatallyfromPapsmears,initiallythroughacustom multiplexPCRplatformtosimultaneouslyamplifySNPsandSTRs,aswelldisease-specificloci,forgenotyping byNGS.Wewillincorporatethelocusforthesicklecellanemia(SCA)pointmutation,whichwillbeexpanded tootherdiseasesinPhaseII.Wewillaccomplishfourmilestones.1.Primerswillbedesignedandtestedwith humangenomicDNAtoamplifySTR,SNPandSCAloci,sequencingPCRproductsbyNGStooptimizetheir amplificationandco-amplificationinsingleplexandmultiplexPCR.2.DNAfromfetalandmaternalcellsisolated byTRIC(N=50),aswellasthecorrespondingnewbornbloodspots(reference),willbeisolatedandcompared bytargetedNGSoftheoptimizedmultiplexPCRproducts.Weexpectampliconstobegeneratedforeachsetof primers.3.STRandSNPhaplotypeswillbeidentified,basedonreaddistributionsintheNGSdata,todetermine whetherfetalDNAdiffersfrommaternalDNA,andisidenticaltothecorrespondingnewbornbloodspotDNA. The fetal fraction will also be determined. 4. DNA from patients carrying a fetus at risk for SCA (N=50) will be analyzed by targeted NGS to compare STR, SNP and SCA haplotypes among fetal, maternal and newborn bloodspot DNA. We expect to demonstrate unique identities for fetal and maternal DNA, identical fetal and newborn haplotypes, and concordance between the SCA haplotype of fetal and newborn DNA. With an estimated annual market potential of $1 billion, the envisioned technology will fill an existing gap in clinical diagnosticsandoutcompeteexistingprenataltestingtechnologies.Ourinitialcommercialproductwilltoenable managementofhighriskpregnancies,andprovidevaluableinformationtophysiciansandpatientsintheprocess ofestablishingfamilies.Specifically,thisinitialproductwillbenefitpregnanciesatriskofhavingachildwithSCA.